Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS). The ENS contains all of the circuitry needed to regulate GI function but it does this in collaboration with information coming from visceral afferents, sympathetic and parasympathetic efferent neurons as well as being sensitive to neuromodulators released from the intestinal epithelium. A rich literature exists demonstrating the importance of these individual inputs for normal GI function, but until recently it has been difficult to investigate the connectome within the ENS or the connectivity between the ENS and its extrinsic inputs that might underlie pathological states. The first step in exploring the use of neuromodulation approaches to treating GI disease is elucidating the intrinsic and extrinsic connectome that allows the ENS to maintain gut homeostasis. To do this we propose to use electrophysiology, imaging, optogenetics, and molecular phenotyping to identify how ENS neurons communicate with each other, extrinsic sensory and autonomic neurons and the gut epithelium. This OT2 application follows successful competition of our OT1 application in response to RFA-RM-15-018 with the goal of developing a comprehensive functional map of neuroanatomy and neurobiology of the enteric neural circuits and associated extrinsic innervation. We will stimulate or monitor specific subsets of neurons based on chemical coding, electrophysiological membrane properties or synaptic components in new mouse models. The goal of generating a predictive functional and anatomical neural circuit map will be accomplished by the following specific aims: 1) Generate a dynamic anatomical map of the ENS circuitry, in genetically modified mice expressing the Ca2+ indicator GCaMP6, ChR2 and/or fluorescent markers; 2) Generate a dynamic anatomical map of ENS intrinsic and extrinsic afferent circuits to determine how input from the epithelium and peripheral nervous system interfaces with the ENS in mice expressing optogenetic actuators/sensors, and 3) determine the molecular signature of mouse and human ENS neurons. Taking all anatomical and functional data together we will generate testable predictive mathematical models of ENS circuitry and its extrinsic innervation.